Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods...Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.展开更多
文摘Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.
